Limits...
Identification of platelet function defects by multi-parameter assessment of thrombus formation.

de Witt SM, Swieringa F, Cavill R, Lamers MM, van Kruchten R, Mastenbroek T, Baaten C, Coort S, Pugh N, Schulz A, Scharrer I, Jurk K, Zieger B, Clemetson KJ, Farndale RW, Heemskerk JW, Cosemans JM - Nat Commun (2014)

Bottom Line: Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3.Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome.We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.

ABSTRACT
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Show MeSH

Related in: MedlinePlus

Measurement of thrombus formation on microspot arrays of platelet-adhesive surfaces.(a) Schematic drawing of the used parallel-plate flow chamber (3 mm width, 50 μm depth) and microspot-coated coverslip. Note the small-angular (11°) chamber inlet and outlet, preventing flow perturbations. (b) Distribution of DiOC6-labelled platelets adhered to consecutive microspots of collagen type I, collagen type III and vWF, after 3.5-min flow of blood at wall shear rate of 1,600 s−1. Given are tile scans of fluorescence images of the full microspots (bar, 100 μm). Black rims are image artifacts due to the tile scanning. Lower panels are enlarged images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109023&req=5

f2: Measurement of thrombus formation on microspot arrays of platelet-adhesive surfaces.(a) Schematic drawing of the used parallel-plate flow chamber (3 mm width, 50 μm depth) and microspot-coated coverslip. Note the small-angular (11°) chamber inlet and outlet, preventing flow perturbations. (b) Distribution of DiOC6-labelled platelets adhered to consecutive microspots of collagen type I, collagen type III and vWF, after 3.5-min flow of blood at wall shear rate of 1,600 s−1. Given are tile scans of fluorescence images of the full microspots (bar, 100 μm). Black rims are image artifacts due to the tile scanning. Lower panels are enlarged images.

Mentions: By coating these proteins/peptides alone or in combinations as a row of three microspots (diameter 1,000 μm, separation 2,000 μm), and mounting the coated coverslips in a standard parallel-plate perfusion chamber38, it was possible to determine the thrombus-forming activity of multiple surfaces at the same time (Fig. 2a). Tile scans performed after perfusion of 3,3′dihexyloxacarbocyanine iodide (DiOC6)-labelled whole blood indicated homogeneous adhesion of platelets to the microspots, either as single cells or in aggregates depending on the surface coating (Fig. 2b). Control experiments further established that the order of coated proteins in microspots did not affect platelet deposition. For instance, microspotting of fibrinogen, fibronectin or collagen I, at upstream or downstream positions, resulted in the same amount of platelet adhesion and aggregation (Supplementary Fig. 1). This pointed to the absence of significant paracrine cross-talk from activated platelets on adjacent microspots.


Identification of platelet function defects by multi-parameter assessment of thrombus formation.

de Witt SM, Swieringa F, Cavill R, Lamers MM, van Kruchten R, Mastenbroek T, Baaten C, Coort S, Pugh N, Schulz A, Scharrer I, Jurk K, Zieger B, Clemetson KJ, Farndale RW, Heemskerk JW, Cosemans JM - Nat Commun (2014)

Measurement of thrombus formation on microspot arrays of platelet-adhesive surfaces.(a) Schematic drawing of the used parallel-plate flow chamber (3 mm width, 50 μm depth) and microspot-coated coverslip. Note the small-angular (11°) chamber inlet and outlet, preventing flow perturbations. (b) Distribution of DiOC6-labelled platelets adhered to consecutive microspots of collagen type I, collagen type III and vWF, after 3.5-min flow of blood at wall shear rate of 1,600 s−1. Given are tile scans of fluorescence images of the full microspots (bar, 100 μm). Black rims are image artifacts due to the tile scanning. Lower panels are enlarged images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109023&req=5

f2: Measurement of thrombus formation on microspot arrays of platelet-adhesive surfaces.(a) Schematic drawing of the used parallel-plate flow chamber (3 mm width, 50 μm depth) and microspot-coated coverslip. Note the small-angular (11°) chamber inlet and outlet, preventing flow perturbations. (b) Distribution of DiOC6-labelled platelets adhered to consecutive microspots of collagen type I, collagen type III and vWF, after 3.5-min flow of blood at wall shear rate of 1,600 s−1. Given are tile scans of fluorescence images of the full microspots (bar, 100 μm). Black rims are image artifacts due to the tile scanning. Lower panels are enlarged images.
Mentions: By coating these proteins/peptides alone or in combinations as a row of three microspots (diameter 1,000 μm, separation 2,000 μm), and mounting the coated coverslips in a standard parallel-plate perfusion chamber38, it was possible to determine the thrombus-forming activity of multiple surfaces at the same time (Fig. 2a). Tile scans performed after perfusion of 3,3′dihexyloxacarbocyanine iodide (DiOC6)-labelled whole blood indicated homogeneous adhesion of platelets to the microspots, either as single cells or in aggregates depending on the surface coating (Fig. 2b). Control experiments further established that the order of coated proteins in microspots did not affect platelet deposition. For instance, microspotting of fibrinogen, fibronectin or collagen I, at upstream or downstream positions, resulted in the same amount of platelet adhesion and aggregation (Supplementary Fig. 1). This pointed to the absence of significant paracrine cross-talk from activated platelets on adjacent microspots.

Bottom Line: Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3.Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome.We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands.

ABSTRACT
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α6β1, αIIbβ3>α2β1>CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Show MeSH
Related in: MedlinePlus